tRNA-derived small RNA (tsRNA) are small RNAs that are derived from the cleavage of tRNA. tRNA fragments of 30-35 nucleotides have been identified in bacteria, fungi, plants, and animals (Lee and Collins, J. Biol. Chem., 280:42744-9 (2005); Haiser et al. Nucleic Acids Res. 36:732-41 (2008); Jochl et al. Nucleic Acids Res. 36:2677-89 (2008); Kawaji et al BMC Genomics 9:157 (2008); Li et al. Nucleic Acids Res. 36:6048-55 (2008); Thompson et al. RNA 14:2095-103 (2008); and Zhang et al. Plant Physiol. 150:378-87 (2009)). There are numerous different tsRNAs in the cell and the functions of how each of these small RNAs interact and function with other cellular components is not completely known.
Mature tRNAs are essential for mRNA translation in their role of transferring amino acids to a growing polypeptide chain. However, tRNA fragments have recently been identified as a source of non-coding RNAs (reviewed in (Martens-Uzunova et al., 2013; Sobala and Hutvágner, 2011)). tRNA fragments are classified into two classes based on their sizes. The longer 30-35 nt RNA species are called tRNA halves and generated by the endonuclease angiogenin. There is growing evidence that tRNA halves are involved in cellular stress response, cell proliferation, and apoptosis.
The other 18-26 nt non-coding RNAs are called small tRNA fragments (tRF) or tRNA-derived small RNA (tsRNA), which have been classified into three groups: 5′tsRNA (tRF-5 or 5′tRF), type I tsRNA (3′tsRNA, tRF-3, or 3′CCAtRF), and type II tsRNA (tRF-1 or 3′U tRF) (Haussecker et al., 2010; Lee et al., 2009). The 5′ and 3′tsRNAs are derived from the 5′ and 3′ end of mature tRNAs, respectively. The 3′tsRNA contains the CCA sequence added to 3′end during tRNA maturation. The tsRNA type II is processed from the 3′ precursor of tRNA, which ends in polyuridine due to termination by RNA polymerase III.
The biogenesis of tsRNAs is not clear. For the generation of type II tsRNAs and 5′tsRNAs there are mixed reports supporting the role of dicer, and one study supporting the role of the tRNA processing enzyme RNaseZ and tRNA 3′-endonuclease, ELAC2, in type II generation (Babiarz et al., 2008; Cole et al., 2009; Haussecker et al., 2010; Lee et al., 2009), while the generation of the 3′tsRNAs (type I) is unlikely related to dicer processing (Babiarz et al., 2008; Li et al., 2012).
The biological role of tsRNAs is not well understood and there have been attempts to establish whether tsRNAs are associated with Ago (Argonaute) proteins, the key component in RISC (RNA-induced silencing complex) (reviewed in (Bartel, 2004; Croce and Calin, 2005; Kim and Kim, 2012; Pederson, 2010)). The evidence for the presence of tsRNA in RISC comes from studies showing that certain tsRNAs can associate with over-expressed Argonaute proteins (Haussecker et al., 2010; Maute et al., 2013). Furthermore, HisGTG and LeuCAG3′tsRNA as well as GlyGCC3′tsRNA have been found to be associated with endogenous Ago2 protein (Li et al., 2012; Maute et al., 2013). In addition, there is some implication that the over-expressed GlyGCC3′tsRNA from a miRNA hairpin or genomic tRNA can reduce endogenous gene expression through base-pairing with complementary target mRNAs in the 3′UTR (Maute et al., 2013). Synthetic S′tsRNAs can inhibit protein translation regardless of their ability to base-pair with complementary target mRNAs, implying that the cellular function of S′tsRNA differs from microRNA (Sobala and Hutvágner, 2013).
tRNA-derived small RNAs are also found in lower organisms. In Tetrahymena, a 18-22 nt fragment of the 3′tRNA is associated with Twi12 (Tetrahymena Piwi12), which is essential for cell growth, and does not have trans-gene silencing activity (Couvillion et al., 2010). Twi12 activates Xrn2 for RNA processing in the nucleus (Couvillion et al., 2012). In Haloferax volcanii, the Val5′tsRNA binds to the ribosome, and a synthetic Val5′tsRNA was shown to inhibit translation (Gebetsberger et al., 2012). All of these various findings suggest that some of these tsRNAs play important roles in various aspects of cellular function.
Unlike the type II tsRNAs, the 5′tsRNAs and 3′tsRNAs (type I tsRNAs) are derived from mature tRNAs making their sequences more highly conserved between species. As reported herein, these tsRNAs play an important role in cell viability. Specifically, when tsRNAs are depleted, cells undergo apoptosis.
Apoptosis is a genetically programmed cellular event that is characterized by well-defined morphological features, such as cell shrinkage, chromatin condensation, nuclear fragmentation, and membrane blebbing. Kerr et al. Br. J Cancer, 26, 239-257 (1972); Wyllie et al. Int. Rev. Cytol., 68, 251-306 (1980). It plays an important role in normal tissue development and homeostasis, and defects in the apoptotic program are thought to contribute to a wide range of human disorders ranging from neurodegenerative and autoimmunity disorders to neoplasms. Thompson, Science, 267, 1456-1462 91995); Mullauer et al. Mutat. Res, 488, 211-231 (2001).
For example, as reported herein, cells in which LeuCAG3′tsRNA is depleted undergo apoptosis by an unusual mechanism that involves tsRNA-mediated depletion of 40S ribosomal subunits. Thus, methods of regulating tsRNAs can be used to regulate apoptosis and control disease.